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- ItemFluorescence Microscopy of the HIV-1 Envelope(Basel : MDPI, 2020) Carravilla, Pablo; Nieva, José L.; Eggeling, ChristianHuman immunodeficiency virus (HIV) infection constitutes a major health and social issue worldwide. HIV infects cells by fusing its envelope with the target cell plasma membrane. This process is mediated by the viral Env glycoprotein and depends on the envelope lipid composition. Fluorescent microscopy has been employed to investigate the envelope properties, and the processes of viral assembly and fusion, but the application of this technique to the study of HIV is still limited by a number of factors, such as the small size of HIV virions or the difficulty to label the envelope components. Here, we review fluorescence imaging studies of the envelope lipids and proteins, focusing on labelling strategies and model systems.
- ItemAutofluorescence lifetime augmented reality as a means for real-time robotic surgery guidance in human patients(Berlin : Nature Publishing, 2019) Gorpas, Dimitris; Phipps, Jennifer E.; Bec, Julien; Ma, Dinglong; Dochow, Sebastian; Yankelevich, Diego R.; Sorger, Jonathan; Popp, Jürgen; Bewley, Arnaud Fassett; Gandour-Edwards, Regina F.; Marcu, Laura; Farwell, D. GregoryDue to loss of tactile feedback the assessment of tumor margins during robotic surgery is based only on visual inspection, which is neither significantly sensitive nor specific. Here we demonstrate time-resolved fluorescence spectroscopy (TRFS) as a novel technique to complement the visual inspection of oral cancers during transoral robotic surgery (TORS) in real-time and without the need for exogenous contrast agents. TRFS enables identification of cancerous tissue by its distinct autofluorescence signature that is associated with the alteration of tissue structure and biochemical profile. A prototype TRFS instrument was integrated synergistically with the da Vinci Surgical robot and the combined system was validated in swine and human patients. Label-free and real-time assessment and visualization of tissue biochemical features during robotic surgery procedure, as demonstrated here, not only has the potential to improve the intraoperative decision making during TORS but also other robotic procedures without modification of conventional clinical protocols.
- ItemLipid Composition but Not Curvature Is the Determinant Factor for the Low Molecular Mobility Observed on the Membrane of Virus-Like Vesicles(Basel : MDPI, 2018) Urbančič, Iztok; Brun, Juliane; Shrestha, Dilip; Waithe, Dominic; Eggeling, Christian; Chojnacki, JakubHuman Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from which it buds out. Previous studies have shown that the HIV-1 envelope is an environment of very low mobility, with the diffusion of incorporated proteins two orders of magnitude slower than in the plasma membrane. One of the reasons for this difference is thought to be the HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing, which has, until now, been difficult to assess experimentally. To further refine the model of the molecular mobility on the HIV-1 surface, we herein investigated the relative importance of membrane composition and curvature in simplified model membrane systems, large unilamellar vesicles (LUVs) of different lipid compositions and sizes (0.1–1 µm), using super-resolution stimulated emission depletion (STED) microscopy-based fluorescence correlation spectroscopy (STED-FCS). Establishing an approach that is also applicable to measurements of molecule dynamics in virus-sized particles, we found, at least for the 0.1–1 µm sized vesicles, that the lipid composition and thus membrane rigidity, but not the curvature, play an important role in the decreased molecular mobility on the vesicles’ surface. This observation suggests that the composition of the envelope rather than the particle geometry contributes to the previously described low mobility of proteins on the HIV-1 surface. Our vesicle-based study thus provides further insight into the dynamic properties of the surface of individual HIV-1 particles, as well as paves the methodological way towards better characterisation of the properties and function of viral lipid envelopes in general.
- ItemAn emerging Panton–Valentine leukocidin-positive CC5-meticillin-resistant Staphylococcus aureus-IVc clone recovered from hospital and community settings over a 17-year period from 12 countries investigated by whole-genome sequencing(Kidlington [u.a.] : Elsevier, 2022) Aloba, B.K.; Kinnevey, P.M.; Monecke, S.; Brennan, G.I.; O'Connell, B.; Blomfeldt, A.; McManus, B.A.; Schneider-Brachert, W.; Tkadlec, J.; Ehricht, R.; Senok, A.; Bartels, M.D.; Coleman, D.C.Background: A novel Panton–Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC)5-MRSA-IVc (‘Sri Lankan’ clone) was recently described from Sri Lanka. Similar isolates caused a recent Irish hospital outbreak. Aim: To investigate the international dissemination and diversity of PVL-positive CC5-MRSA-IVc isolates from hospital and community settings using whole-genome sequencing (WGS). Methods: Core-genome single nucleotide polymorphism (cgSNP) analysis, core-genome multi-locus sequence typing (cgMLST) and microarray-based detection of antimicrobial-resistance and virulence genes were used to investigate PVL-positive CC5-MRSA-IVc (N = 214 including 46 ‘Sri Lankan’ clone) from hospital and community settings in 12 countries over 17 years. Comparators included 29 PVL-positive and 23 PVL-negative CC5/ST5-MRSA-I/II/IVa/IVc/IVg/V. Results: Maximum-likelihood cgSNP analysis grouped 209/214 (97.7%) CC5-MRSA-IVc into Clade I; average of 110 cgSNPs between isolates. Clade III contained the five remaining CC5-MRSA-IVc; average of 92 cgSNPs between isolates. Clade II contained seven PVL-positive CC5-MRSA-IVa comparators, whereas the remaining 45 comparators formed an outlier group. Minimum-spanning cgMLST analysis revealed a comparably low average of 57 allelic differences between all CC5/ST5-MRSA-IVc. All 214 CC5/ST5-MRSA-IVc were identified as ‘Sri Lankan’ clone, predominantly spa type t002 (186/214) with low population diversity and harboured a similar range of virulence genes and variable antimicrobial-resistance genes. All 214 Sri Lankan clone isolates and Clade II comparators harboured a 9616-bp chromosomal PVL-encoding phage remnant, suggesting both arose from a PVL-positive meticillin-susceptible ancestor. Over half of Sri Lankan clone isolates were from infections (142/214), and where detailed metadata were available (168/214), most were community associated (85/168). Conclusions: Stable chromosomal retention of pvl may facilitate Sri-Lankan clone dissemination.
- ItemMultiple distinct outbreaks of Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus in Ireland investigated by whole-genome sequencing(Kidlington [u.a.] : Elsevier, 2021) McManus, B.A.; Aloba, B.K.; Earls, M.R.; Brennan, G.I.; O'Connell, B.; Monecke, S.; Ehricht, R.; Shore, A.C.; Coleman, D.C.Background Panton–Valentine leucocidin (PVL)-positive community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) is increasingly associated with infection outbreaks. Aim To investigate multiple suspected PVL-positive CA-MRSA outbreaks using whole-genome sequencing (WGS). Methods Forty-six suspected outbreak-associated isolates from 36 individuals at three separate Irish hospitals (H1–H3) and from separate incidents involving separate families associated with H2 were investigated by whole-genome multi-locus sequence typing (wgMLST). Findings Two clusters (CH1 and CH2) consisting of 8/10 and 6/6 PVL-positive t008 ST8-MRSA-IVa isolates from H1 and H2, respectively, were identified. Within each cluster, neighbouring isolates were separated by ≤5 allelic differences; however, ≥73 allelic differences were identified between the clusters, indicating two independent outbreaks. Isolates from the H3 maternity unit formed two clusters (CH3–SCI and CH3–SCII) composed of four PVL-negative t4667 ST5-MRSA-V and 14 PVL-positive t002 ST5-MRSA-IVc isolates, respectively. Within clusters, neighbouring isolates were separated by ≤24 allelic differences, whereas both clusters were separated by 1822 allelic differences, indicating two distinct H3 outbreaks. Eight PVL-positive t127 ST1-MRSA-V+fus and three PVL-negative t267 ST97-MRSA-V+fus isolates from two distinct H2-associated families FC1 (N = 4) and FC2 (N = 7) formed three separate clusters (FC1 (t127), FC2 (t127) and FC2 (t267)). Neighbouring isolates within clusters were closely related and exhibited ≤7 allelic differences. Intrafamilial transmission was apparent, but the detection of ≥48 allelic differences between clusters indicated no interfamilial transmission. Conclusion The frequent importation of PVL-positive CA-MRSA into healthcare settings, transmission and association with outbreaks is a serious ongoing concern. WGS is a highly discriminatory, informative method for deciphering such outbreaks conclusively.
- ItemSuper-Resolution STED Microscopy-Based Mobility Studies of the Viral Env Protein at HIV-1 Assembly Sites of Fully Infected T-Cells(Basel : MDPI, 2021) Chojnacki, Jakub; Eggeling, ChristianThe ongoing threat of human immunodeficiency virus (HIV-1) requires continued, detailed investigations of its replication cycle, especially when combined with the most physiologically relevant, fully infectious model systems. Here, we demonstrate the application of the combination of stimulated emission depletion (STED) super-resolution microscopy with beam-scanning fluorescence correlation spectroscopy (sSTED-FCS) as a powerful tool for the interrogation of the molecular dynamics of HIV-1 virus assembly on the cell plasma membrane in the context of a fully infectious virus. In this process, HIV-1 envelope glycoprotein (Env) becomes incorporated into the assembling virus by interacting with the nascent Gag structural protein lattice. Molecular dynamics measurements at these distinct cell surface sites require a guiding strategy, for which we have used a two-colour implementation of sSTED-FCS to simultaneously target individual HIV-1 assembly sites via the aggregated Gag signal. We then compare the molecular mobility of Env proteins at the inside and outside of the virus assembly area. Env mobility was shown to be highly reduced at the assembly sites, highlighting the distinct trapping of Env as well as the usefulness of our methodological approach to study the molecular mobility of specifically targeted sites at the plasma membrane, even under high-biosafety conditions.
- ItemStructural insights into heme binding to IL-36α proinflammatory cytokine(Berlin : Nature Publishing, 2019) Wißbrock, Amelie; Goradia, Nishit; Kumar, Amit; Paul George, Ajay Abisheck; Kühl, Toni; Bellstedt, Peter; Ramachandran, Ramadurai; Hoffmann, Patrick; Galler, Kerstin; Popp, Jürgen; Neugebauer, Ute; Hampel, Kornelia; Zimmermann, Bastian; Adam, Susanne; Wiendl, Maximilian; Krönke, Gerhard; Hamza, Iqbal; Heinemann, Stefan H.; Frey, Silke; Hueber, Axel J.; Ohlenschläger, Oliver; Imhof, DianaCytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.
- ItemIn Vitro Selection of Specific DNA Aptamers Against the Anti-Coagulant Dabigatran Etexilate(Berlin : Nature Publishing, 2018) Aljohani, Maher M; Chinnappan, Raja; Eissa, Shimaa; Alsager, Omar A; Weber, Karina; Cialla-May, Dana; Popp, Jürgen; Zourob, MohammedDabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8–208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.Dabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8–208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.
- ItemMolecular Characterization and Comparative Genomics of Clinical Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains in Sweden(Berlin : Nature Publishing, 2019) Bai, Xiangning; Zhang, Ji; Ambikan, Anoop; Jernberg, Cecilia; Ehricht, Ralf; Scheutz, Flemming; Xiong, Yanwen; Matussek, AndreasHybrid E. coli pathotypes are representing emerging public health threats with enhanced virulence from different pathotypes. Hybrids of Shiga toxin-producing and enterotoxigenic E. coli (STEC/ETEC) have been reported to be associated with diarrheal disease and hemolytic uremic syndrome (HUS) in humans. Here, we identified and characterized four clinical STEC/ETEC hybrids from diarrheal patients with or without fever or abdominal pain and healthy contact in Sweden. Rare stx2 subtypes were present in STEC/ETEC hybrids. Stx2 production was detectable in stx2a and stx2e containing strains. Different copies of ETEC virulence marker, sta gene, were found in two hybrids. Three sta subtypes, namely, sta1, sta4 and sta5 were designated, with sta4 being predominant. The hybrids represented diverse and rare serotypes (O15:H16, O187:H28, O100:H30, and O136:H12). Genome-wide phylogeny revealed that these hybrids exhibited close relatedness with certain ETEC, STEC/ETEC hybrid and commensal E. coli strains, implying the potential acquisition of Stx-phages or/and ETEC virulence genes in the emergence of STEC/ETEC hybrids. Given the emergence and public health significance of hybrid pathotypes, a broader range of virulence markers should be considered in the E. coli pathotypes diagnostics, and targeted follow up of cases is suggested to better understand the hybrid infection.
- ItemEx vivo Hyperspectral Autofluorescence Imaging and Localization of Fluorophores in Human Eyes with Age-Related Macular Degeneration(Basel : MDPI, 2018) Mohammed, Taariq; Tong, Yuehong; Agee, Julia; Challa, Nayanika; Heintzmann, Rainer; Hammer, Martin; Curcio , Christine A.; Ach, Thomas; Ablonczy, Zsolt; Smith, R. TheodoreTo characterize fluorophore signals from drusen and retinal pigment epithelium (RPE) and their changes in age related macular degeneration (AMD), the authors describe advances in ex vivo hyperspectral autofluorescence (AF) imaging of human eye tissue. Ten RPE flatmounts from eyes with AMD and 10 from eyes without AMD underwent 40× hyperspectral AF microscopic imaging. The number of excitation wavelengths tested was initially two (436 nm and 480 nm), then increased to three (436 nm, 480 nm, and 505 nm). Emission spectra were collected at 10 nm intervals from 420 nm to 720 nm. Non-negative matrix factorization (NMF) algorithms decomposed the hyperspectral images into individual emission spectra and their spatial abundances. These include three distinguishable spectra for RPE fluorophores (S1, S2, and S3) in both AMD and non-AMD eyes, a spectrum for drusen (SDr) only in AMD eyes, and a Bruch’s membrane spectrum that was detectable in normal eyes. Simultaneous analysis of datacubes excited atthree excitation wavelengths revealed more detailed spatial localization of the RPE spectra and SDr within drusen than exciting only at two wavelengths. Within AMD and non-AMD groups, two different NMF initialization methods were tested on each group and converged to qualitatively similar spectra. In AMD, the peaks of the SDr at ~510 nm (436 nm excitation) were particularly consistent. Between AMD and non-AMD groups, corresponding spectra in common, S1, S2, and S3, also had similar peak locations and shapes, but with some differences and further characterization warranted.